Mammalian ras proteins bind guanine nucleotides and have been implicated in the malignant cell transformation, as well as in the control of cellular proliferation and differentiation. We have recently identified and purified ras guanine nucleotide exchange factor (rGEF) which is capable of stimulating the GDP/GTP exchange reaction of ras proteins. Studies were performed to establish the physiological role of rGEF in ras-mediated signal transduction. In order to conduct the molecular biological studies of rGEF functions, the molecular cloning of cDNA encoding rGEF is in progress. For the immunological screening of the cDNA library, we are producing the monoclonal antibodies against rGEF. The domain of ras proteins involved in the interaction with rGEF was also investigated by the mutational analysis. Our data indicated that 58-68 amino acid residues of ras protein may be responsible for the interaction. In another approach, we analyzed the response of ras activity to the growth signal. The active ras-GTP complex was accumulated in serum-stimulated NIH 3T3 cells. It is possible that rGEF and/or GAP play important regulatory roles in ras-mediated cell proliferation/differentiation. Also, we have developed an in vitro reconstitution system. These studies will provide a further insight into the regulatory mechanism of ras activity in transmembrane signal transduction pathway.